HIV-encoded chemoattractant

ABSTRACT

The combination of HIV proteins Tat and Nef is chemotactic for CD4+ cells. Utilizing the capacity of Tat and Nef to modulate CD4+ cell trafficking and infiltration, the invention provides various treatment modes for individuals infected with HIV. The invention further provides treatment modes for other localized diseases by controlling CD4+ cell trafficking and infiltration. In particular, the invention provides methodology for promoting CD4+ cell chemotaxis to a localized site of infection as a means of augmenting the efficacy of extant chemotherapeutic methods. The invention further provides methodology for diverting CD4+ cell infiltration from a localized site where the presence of CD4+ cells is detrimental to the clinical outcome, by providing a composition comprising Tat and Nef at a distinct site, such as blood, within the individual where the accumulation of CD4+ cells is less detrimental.

This application is a divisional of U.S. application Ser. No.10/274,361, filed Oct. 21, 2002, now U.S. Pat. No. 6,878,685 which is acontinuation of U.S. application Ser. No. 09/479,596 filed Jan. 7, 2000,now abandoned which claims priority to U.S. Provisional Application Ser.No. 60/116,929 filed Jan. 22, 1999.

The United States government has certain rights to this invention as aresult of federal funding from National Institute of Health grantDE12161 and NIAID A140040.

BACKGROUND OF THE INVENTION

The recruitment and trafficking of CD4+ cells in the body is modulatedby chemoattractant proteins, typically produced in response toinflammatory stimuli. Reviewed in Taub (1996). Directed CD4+ cellmovement up concentration gradients of chemoattractants results intrafficking of CD4+ cells out of the circulation, through the vascularendothelium, and into peripheral tissue to the site of highest localizedconcentration of chemoattractants. By secreting their ownchemoatractants, CD4+ cells may direct the subsequent accumulation ofvarious other cells types involved in host defense mechanisms, such asneutrophils. While the localized. accumulation of CD4+ cells may beimportant for defense against infection, accumulation of CD4+ cells canalso be part of a chain of events resulting in an unwanted inflammatoryresponse.

For example, infiltration and accumulation of CD4+ cells in therespiratory tract helps mediate clearance of Cryptococcus neoformansinfections, which otherwise will disseminate to cause meningitis.Huffnagle et al. (1995). By contrast, uncontrolled infiltration andactivation of CD4+ cells in response to bacterial antigens appears to bethe underlying cause of inflammatory bowel disease. Van Deventer et al.(1997). Localized CD4+ cell accumulation has been implicatedspecifically in response to infections and inflammatory conditions,including inflammatory bowl disease, bacterial infection, viralinfection, atherosclerosis, asthma, graft versus host disease (GVHD),endotoxemia, uveoretinitis, psoriasis, and granulomatous diseases.Center (1996); Jinquan et al. (1995); Taub (1996); Van de Kerkhof et al.(1996). See, in general, Staniford (1997).

Another disease associated with CD4+ cell physiology is HIV infection. Adefining feature of progression of HIV infection to Acquired ImmuneDeficiency Syndrome (AIDS) is a decline in the number of CD4+ cells inthe infected individual. Reviewed in Levy (1993). Disease progressionalso strongly correlates with a switch from non-syncytium-forming (NSI)HIV variants to syncytium-forming (SI) variants within infectedindividuals. Richman et al. (1994); Miedema (1992). For example, arecent study found that the adenoid tissue of 13 of 13 asymptomaticindividuals contained syncytia formed apparently from fusion ofdendritic cells and T cells. Frankel et al. (1996).

Recent studies have provided a potential link between CD4+ cell loss andsyncytium formation by implicating CD4+ cell chemotaxis in thepropagation of syncytia. Syncytia have been found to release CD4+ cellchemoattractants, potentially providing a mechanism by which CD4+ cellscan be recruited throughout the body to their demise by incorporationinto short-lived syncytia. Shutt et al. (1998).

Syncytia are multinucleated conglomerates of HIV-infected cells havingup to many thousands of times the volume of a single cell. They areformed when the virally encoded glycoprotein gp120 on the surface ofinfected cells interacts with the CD4 receptor of uninfected cells toinitiate cell fusion. Kowolski et al. (1987); McDougal et al. (1986);Lifson et al. (1986a,b). Syncytia also are phagocytotic and may engulfentire CD4+ cells. Sylwester et al. (1995). In vitro studies haveindicated that up to 90% of T cell death is accounted for by theirincorporation into syncytia. Vast quantities of HIV are released uponthe death of syncytia, which infect non-fused CD4+ cells, thus creatinga self-perpetuating cycle of infection and CD4+ cell death. Sylwester etal. (1997).

In addition to perpetuating HIV infection, syncytia can directly causetissue destruction by virtue of motile properties retained fromconstituent lymphocyte cells. Soll, 1997. Syncytia move by extending andretracting giant pseudopodia and filopodia, which penetrate and disruptcollagen and endothelial tissue substrates. Sylwester et al. (1998). Theability of syncytia to degrade and extravasate through endothelialtissue may be related to the destruction of lymph node architecture andthe leakiness of blood vessels in an individual bearing thesyncytium-inducing variant of HIV. This property may also account forthe apparent absence of syncytia in blood vessels, since these largecells would likely become stuck in capillaries, where they could thenextravasate into the periphery.

In videorecordings of fields of single cells and syncytia in the act offusing in vitro, it was apparent that single cells and small syncytiamoved in a persistent and directed fashion towards large syncytia,suggesting that the latter released a T cell chemoattractant. To confirmthis hypothesis, a specialized single-cell chemotaxis chamber was usedto discriminate chemokinesis from chemotaxis. Shutt et al. (1998).Chemokinesis is accelerated, non-vectorial movement, while chemotaxis isdirected movement of cells up a concentration gradient of achemoattractant. The microfilter assay, first introduced by Boyden(1962), was not used because it cannot unambiguously distinguishchemotaxis form chemokinesis, even when the appropriate corrections areperformed. Wilkinson (1988); Zigmond (1978); Zigmond et al. (1973);Rhodes (1982); Shutt et al. (1998).

Syncytia release two chemotactic components into the supernatant, havingapproximate molecular weights of 30 and 120 kDa. Shutt et al. (1998).Virally encoded glycoprotein gp120 is released into the medium byHIV-infected cells, implicating the 120 kDa chemoattractant as gp120.Gelderblom et al. (1985); Schneider et al. (1986). This was confirmed bythe ability of anti-gp120 antibody to block the high molecular weightchemoattractant, and by the ability of purified gp120 to attract Tcells. Shutt et al. (1998).

The identity of the lower molecular weight component remained unknown.HIV virus encodes several low molecular weight proteins, including Rev,p24, Nef (27 kDa), a negative regulator of viral replication, and Tat(15.5 kDa), an activator of viral gene expression, but there . was nosuggestion in the art that these proteins function as chemoattractants,with the possible exception of Tat. Tat induces the migration ofmonocyte-derived dendritic cells and monocytes across a membrane in aBoyden chamber in a concentration-dependent manner. Benelli et al.(1998). While these authors concluded that Tat acted as a dendritic cellchemoattractant, the absence of critical controls suggested by Zigmondet al. (1973) for the microfilter assay complicates the distinctionbetween a chemotactic and a chemokinetic effect.

There have been, however, no reports of either chemotactic orchemokinetic activity by the other low molecular weight HIV proteins.Although interactions of Nef with various cellular proteins have beencharacterized, the known interactions are not suggestive of chemotacticor chemokinetic activity. The activity of a putative CD4+ cellchemoattractant, interleukin-16, is thought to require coupling betweenCD4 and the cellular kinase p56^(Ick) Ryan et al. (1995). However,intracellular Nef expression has been demonstrated to reduce cellsurface expression of CD4. Gratton et al. (1996). Further, Nefapparently disrupts the interaction between CD4 and p56^(Ick), eitherdirectly or, more likely, indirectly through other proteins recruited byNef. Otake et al. (1994); Benichou et al. (1994).

Thus, there is a need in the art to modulate CD4+ cell chemotaxis. Sucha capability will enhance treatment of a disease associated withlocalized CD4+ cell accumulation. It is desirable to direct CD4+chemotaxis to a localized site of infection in circumstances where CD4+cell intervention is desired for a favorable clinical outcome. Likewise,it is desirable to prevent CD4+ cell chemotaxis at sites where thisaccumulation leads to undesired inflammatory reactions. Alternately, itis desirable to redirect CD4+ cell accumulation to sites where theiraccumulation will be less detrimental.

Modulation of CD4+ cell chemotaxis would be desirable in treatment ofHIV infection as a means of inhibiting the syncytium-induced chemotaxisof CD4+ cells out of circulation and into peripheral or lymphoid tissue,where they may fuse with or be engulfed and destroyed by syncytia. Byinhibiting syncytium-mediated T cell death, the progression of HIVinfection into AIDS may be retarded.

SUMMARY OF THE INVENTION

The invention provides a method for modulating CD4+ cell chemotaxis.This is accomplished by a composition comprising a combination of theHIV proteins Tat and Nef. By themselves, these proteins have achemokinetic effect on CD4+ cells. Surprisingly, in combination, theyare chemotactic for CD4+ cells. Thus, the chemoattractant activity ofthe apparent 30 kDa molecular weight syncytium-conditioned mediumcomponent is actually due to the combined effects of Nef and Tat.

CD4+ cell chemotaxis may be stimulated by administering a compositioncomprising Tat and Nef. This composition may be locally administered toa site of infection to promote CD4+ cell chemotaxis where the presenceof CD4+ cells is desirable to the clinical outcome. Alternately,peripheral administration of the composition may be used to divert CD4+cell accumulation from sites where their presence is detrimental.Preferably, the composition may be administered along with achemotherapeutic agent. In this embodiment, the local infiltration ofCD4+ cells stimulated by Tat and Nef augments the therapeutic value ofthe chemotherapeutic agent.

In a preferred embodiment, the composition is useful for the treatmentof HIV infection. Extravasation of CD4+ cells into peripheral tissue maybe inhibited by systemically administering the inventive composition.High systemic concentrations of Tat and Nef may mask or obscuregradients of these same molecules originating from syncytia inperipheral tissue. Because chemotaxis is directed toward higherconcentrations of the chemoattractant, systemic administration of Tatand Nef would inhibit the migration of CD4+ cells into the periphery andwould consequently inhibit their recruitment into syncytia.

Accordingly, it is an object of the invention to provide apharmacological composition comprising Tat and Nef proteins, or afunctional fragment or derivative thereof, in an amount effective toinduce CD4+ cell chemotaxis. In a preferred embodiment, Tat and Nef areadministered as purified proteins in a pharmacologically acceptablediluent, carrier, stabilizer, or excipient. In a more preferredembodiment, Tat and Nef are administered in the presence of anadditional chemotherapeutic agent, which is delivered in an effectiveamount to treat the infection. In another preferred embodiment, Tat andNef are produced recombinantly.

The invention also provides a method of treating a disease, comprisingadministering a composition comprising Tat and Nef to promote CD4+ cellchemotaxis and accumulation at the site of administration, where theaccumulation of CD4+ cells is desirable to the therapeutic outcome of adisease. In a preferred embodiment, the site of administration is at alocalized focus of infection, and a chemotherapeutic agent isco-administered with Tat and Nef, so that the infiltration of CD4+ cellsat the focus of infection augments the effect of the chemotherapeuticagent.

The invention also provides a method of treating a disease, comprisingadministering a composition comprising Tat and Nef to alleviate alocalized, detrimental infiltration of CD4+ cells occurring inassociation with a disease state. The site of administration of thecomposition comprising Tat and Nef may be in a different tissue ororgan, for example, to promote T cell accumulation at a site lessdetrimental that associated with the disease state. Alternately,administration may be systemic to cause CD4+ cells to leave peripheraltissue.

It is another object of the invention to treat individuals infected withHIV by inhibiting CD4+ cell extravasation with a systemicallyadministered composition comprising Tat and Nef. It is another object ofthe invention to antagonize the chemotactic activity of the combinationof Tat and Nef in an individual infected with HIV by providingantibodies to either or both of these proteins.

These and other objects of the invention are accomplished by theembodiments of the invention described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the calibration curve determined by measuring the responsetime of known CD4+ chemoattractants, using the single-cell chemotaxischamber disclosed by Shutt et al. (1998). Response times determined forpeak 1 and peak 2 of syncytia-conditioned culture medium are indicated.

FIG. 1B shows the proportion of chemotactically responsive cells (CP+)as a function of time following establishment of a gradient in thesingle-cell chemotaxis chamber. Peaks 1 and 2 of syncytia-conditionedculture medium are indicated.

FIGS. 2A-D show representative time-dependent tracks of CD4+ cellsexposed to the indicated gradients. The direction of the concentrationgradient is indicated by the heavy arrows.

FIG. 3 shows representative time-dependent cellular tracks in thepresence of a gradient of purified Tat and Nef.

FIGS. 4A-D show CP+ as a function of time for the indicated mediacomponents. FIG. 4D indicates the effect on chemotactic activity ofmedia supplemented with syncytia-conditioned media in response to theaddition of an antibody that binds Nef, “αNef.”

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The Combination of Tat and Nef is a CD4+ Cell Chemoattractant

For the first time, it is possible to modulate CD4+ chemotaxis bypromoting or antagonizing the chemotactic activity of the combination ofTat and Nef. Controlling CD4+ cell trafficking and infiltration in anindividual opens many therapeutic possibilities, especially in treatingdiseases having a localized nature, as well as in providing treatmentmodes for individuals infected with HIV. In view of the activity of Tatand Nef in modulating localized accumulation of CD4+ cells, theinvention provides methodology for promoting CD4+ cell chemotaxis to alocalized site of infection as a means of augmenting the efficacy ofextant chemotherapeutic methods. The invention further providesmethodology for diverting CD4+ cell infiltration from a localized sitewhere the presence of CD4+ cells is detrimental to the clinical outcome,by providing a composition comprising Tat and Nef at a distinct site,such as blood, within the individual where the accumulation of CD4+cells is less detrimental.

Preferably, the present composition is administered in the presence ofknown chemotherapeutic agents as a means of augmenting their efficacy.For instance, a localized infection may be treated by the non-systemicinjection of a composition comprising Tat and Nef at the site ofinfection in the presence of an antibiotic. The promotion of CD4+ cellsto the site of infection will aid in the body's defense against theinfection, enhancing the effect of the antibiotics. “CD4+ cell” means alymphocyte having cell surface CD4 molecules, as evidenced by binding ofan antibody specific for CD4. An example of a suitable antibody is themonoclonal antibody OKT4 (Ortho Diagnostics, Piscataway, N.J.).

In Vitro Assay

An in vitro assay for determining the chemotactic activity of a testmolecule is described in the Examples, below. This test was applied tovarious low molecular weight HIV proteins to determine which mimic theactivity of the 30 kDa component of syncytium-conditioned media. Thechemotactic activity of Rev, p24, Nef and Tat gradients was tested. Revand p24 were neither chemotactic nor chemokinetic for CD4+ cells.Neither Nef alone nor Tat alone was chemotactic, but both werechemokinetic for these cells. Tat and Nef both stimulated single cellmotility when added separately, resulting in a 50% increase in averageinstantaneous velocity in both cases.

Surprisingly, when Tat and Nef are added to the source well of thesingle-cell chemotaxis chamber, they function in combination as achemoattractant. Antibodies to either Nef or Tat block the low molecularweight chemotactic activity, but not the chemokinetic activity, of mediaconditioned by HIV-induced syncytia. Thus, the low molecular weightchemotactic activity of syncytia-conditioned culture media comprises agradient of Tat and Nef.

This activity of Tat and Nef is not the result of synergistic effects ofthe two molecules on chemotaxis, because one protein without the otheris incapable of causing CD4+ cell chemotaxis. The requirement for bothproteins for chemoattractant activity is believed to be an unusual, ifnot unprecedented, observation. Without being limited by theory, thedata suggest that the two chemoattractants do not form a complex, suchas a heterodimer, because this would affect their effective molecularweight during the formation of a chemotactic gradient. The molecularevents involved in chemotaxis are complex, involving recognition of thechemotactic gradient, transmission of this recognition within the cell,and the actual generation of cellular motion. The precise eventsassociated with chemotaxis in response to Tat and Nef are not fullyunderstood. Tat and Nef may interact with the same or different cellularreceptors, and the consequent cellular signaling pathway may convergeupon a common intracellular target. The cellular receptor(s) thatmediates chemotaxis may be the same or different from the receptor(s)that mediate chemokinetic stimulation.

The in vitro assay disclosed herein provides a preferred, routine meansof assaying compounds for chemotactic activity for various cell types.The invention envisions the use of functional fragments and derivativesof Tat and Nef. “Functional fragments and derivatives” of Tat and Nefare defined as fragments and derivatives of these proteins that retainchemotactic activity toward CD4+ cells when used in combination.Functional activity may be assayed in the in vitro chemotaxis assaydisclosed in the Examples. Fragments and derivatives of Tat and Nef maybe produced by genetic engineering methods well known in the art, andexpressed by the methods disclosed below.

In Vivo Assays

In addition to the in vitro assay described in detail below, animalmodels may be used to detect CD4+ cell chemoattractants. Like the Boydenassay, these in vivo assays may be hampered by the inability todiscriminate between chemokinetic and chemotactic activities.Accordingly, the chemotactic activity of test components preferably isconfirmed first by the in vitro assay described below before they aretested in vivo.

In vivo assays described in the art suggest the effectiveness oflocalized injection of putative T cell chemoattractants in promoting Tcell extravasation and accumulation in peripheral tissue. Taub (1996).For example, localized CD4+ cell accumulation in vivo has been seenfollowing injection of monocyte chemotactic proteins-1 (MCP-1), -2, or-3 and the cytokine RANTES (Regulated on Activation, Normal T CellExpressed and Secreted). Taub et al. (1995); Murphy et al. (1994). Usinganimal models, the preferred routes of administration of the inventivecomposition may be determined. Animal models will also be useful indetermining physiological doses of Tat and Nef effective to promotelocalized accumulation of CD4+ cells, as well as the optimal carriers,excipients, etc., for administration of the present composition.

Methods of Treating HIV Infection

Systemic infusion of a pharmaceutical composition comprising Tat and Nefcan be useful in treating an individual infected with HIV. Peripherallylocated syncytia are expected to cause extravasation of CD4+ cells inresponse to the syncytium-induced concentration gradient of Tat and Nef.Recognition of a chemotactic gradient is required before leukocytesadhere to endothelial tissue and migrate through this tissue. Reviewedin Proost et al. (1996) and Jinquan et al. (1995). High systemicconcentrations of Tat and Nef are expected to effectively mask achemotactic gradient originating from peripheral tissue, therebyblocking the first, requisite step in leukocyte extravasation. In thismanner, Tat and Nef may inhibit CD4+ cell extravasation and recruitmentinto syncytia. In light of the known chemotactic activity of gp120, thepresent pharmaceutical composition will be supplemented with aneutraizing antibody to gp120.

Preferably, systemic concentrations of Tat and Nef will be high enoughto establish a chemical gradient sufficient to draw CD4+ cells out oflymph nodes and other peripheral tissue, increasing the number ofcirculating CD4+ cells. Localization of T cells in lymphoid tissue maycontribute to the spread of HIV, because lymphoid tissue acts as areservoir for HIV in an infected individual. Transmission of HIV mayoccur through routes other than syncytium formation, such ascell-to-cell transmission. Levy (1993). Accordingly, it is expected thatdecreasing the number of CD4+ cells in peripheral tissues, especiallylymphoid tissue, will decrease viral burden by preventing T cellinfection.

During the initial infection with HIV, plasma levels of HIV rise andCD4+ cell numbers fall. After a period of months, however, CD4+ cellnumbers rebound and plasma HIV levels subside. The infection at thispoint is asymptomatic and is associated with a gradual decline in thenumber of CD4+ cells. Even though the rate of HIV replication is lowduring this period of “cellular latency,” HIV infection is maintained bythe integration of HIV genomes into the DNA of target cells. Thedevelopment of AIDS follows this period of cellular latency. The onsetof AIDS is correlated with the switch from NSI to SI HIV variants, asdescribed above, and the rapid fall of CD4+ cell counts.

Nef and Tat have been implicated as factors possibly involved inregulating cellular latency. While there is some evidence that Nef maymaintain cellular latency, Tat, in fact, may have the opposite effect.Reviewed in Levy (1993). Thus, it is possible that infusion of Tat andNef by themselves into an HIV infected individual may activate latentlyinfected cells, promoting viral replication and the onset of AIDS.Accordingly, the present composition is preferably administered inconjunction with chemotherapeutic agents effective to inhibit HIVreplication. In a preferred embodiment, HIV replication is inhibited byco-administration of the present composition with highly. activeanti-retroviral therapy (HAART) as described in Berger et al. (1998),herein incorporated by reference.

Cellular latency persists only for the life time of the cells harboringthe HIV genome. One strategy for HIV chemotherapy has been todeliberately activate HIV replication in latently infected cells toexpose HIV to a battery of existing chemotherapeutic agents effective atinhibiting viral replication. See U.S. Pat. No. 5,747,526 for a generaldiscussion of known HIV chemotherapies. The desired result of thisapproach is the depletion of the reservoir of HIV in latently infectedcells. The combination of IL-2, IL-6, and tumor necrosis factor-α(TNF-α) is preferred for activating latently infected CD4+ cells. Chunet al. (1998).

Because the administration of Tat and Nef may activate latent cells, itmay be possible to use these compounds to deplete the reservoir of HIVas described above. Accordingly, in one embodiment of the invention, Tatand Nef are administered systemically, either as a steady infusion or asa bolus, to an individual latently infected with HIV in order toactivate latently infected cells, wherein Tat and Nef are addedconcurrently with chemotherapeutic agents effective at inhibiting HIVreplication. In a preferred embodiment, Tat and Nef are administered inassociation with HAART.

Antibodies Against Tat and Nef

The present invention envisions the use of antibodies directed againstTat and Nef as antagonists of chemotaxis, in combination with aneutralizing antibody directed against gp120. Antibodies that recognizethese proteins are disclosed in US Patent No. 5,606,026, for example,herein incorporated by reference. The invention provides a method foradministering the antibodies to inhibit the chemotactic activity of Tator Nef and gp120 by reducing their systemic concentration. In apreferred embodiment, neutralizing antibodies directed against Tat andNef and gp120 are administered in combination with compounds known toactivate latently infected cells, such as IL-1, and chemotherapeuticagents effective to inhibit viral replication. In this embodiment,anti-Tat and anti-Nef antibodies, in combination with anti-gp120antibodies, reduce the cell-to-cell transmission of HIV via syncytia,while IL-1 activates latently infected cells, and HAART suppressesproduction of infectious HIV.

Antibodies against Tat and/or Nef may be raised by using Tat and/or Nefas vaccine components in the form of intact proteins, or as antigenicfragments. In another embodiment, antibodies against Tat and/or Nef maybe raised intracellularly using the procedures disclosed, for example,in U.S. Pat. No. 5,851,829 or Chen et al. (1994).

Vaccine Supplementation with Tat and Nef

Vaccine efficacy may be boosted by the recruitment of CD4+ cells to thesite of vaccine injection. Accordingly, the present invention provides amethod of boosting vaccine efficacy by providing Tat and Nef ascomponents of a vaccine. The chemotactic activity of Tat and Nef willprovoke accumulation of CD4+ cells to the injection site.

CD4+ cells recruited from the circulation contain naive Th0 cells, whichdifferentiate into Th1 and Th2 cells that mediate cellular and humoralimmune reaction, respectively. The pathway of Th0 cell differentiationhas been shown to be effected by particular cytokines. Accordingly, theaddition of these cytokines as additional vaccine components may enhancethe effect of Tat and Nef by biasing immunity against the antigen aseither cellular immunity or humoral imnmunity. Cellular immunity isdesirable for treatment of intracellular parasitic infection.Differentiation of ThO cells into Th1 cells may be stimulated by aneutralizing antibody directed against interleukin4 (α-IL4) or by theaddition of IL-12. Alternately, humoral immunity is desirable for thetreatment of infection by extracellular agents, such as nematodes. Th2cell differentiation may be stimulated by IL-4 or α-interferon γ. Sheret al. (1992); Clerici et al. (1993); Desmedt et al. (1998);Silva-Teixeira et al. (1998).

Methods of Treating Disease States Comprising Chemotaxis of CD4+ Cells

CD4+ cell infiltration in response to inflammatory stimuli is involvedin a large number of disease states. These diseases include, but are notlimited to, inflammatory bowl disease, bacterial infection, viralinfection, atherosclerosis, asthma, graft versus host disease (GVHD),endotoxemia, uveoretinitis, granulomatous diseases, and psoriasis.Center (1996); Jinquan et al. (1995); Taub (1996); Van de Kerkhof et al.(1996).

The present invention provides for provoking CD4+ cell infiltration andaccumulation at peripheral sites where the accumulation of CD4+ cells isdesirable. Such sites may be localized sites of infection, for example.The present composition is preferably administered in conjunction withantibiotics to promote treatment of the infection. For example, toprovide CD4+ cell accumulation in the respiratory tract to promoteclearance of Cryptococcus neoformans infections, the present compositionmay be administered as an aerosol to the afflicted area. Huffnagle etal. (1995).

When CD4+ cells infiltration is undesirable at a particular site, suchas in inflammatory diseases, the present composition may be used todivert CD4+ cells to other areas. For example, the present compositionmay be added systemically in sufficient amounts to cause CD4+ cells toenter the circulation, thereby lowering the peripheral counts of thesecells. Alternately, the present composition may be administered to adifferent peripheral location, such as a different organ, less likelycause inflammatory complications for the patient, causing a diversion ofCD4+ cells from the site of inflammation.

The invention envisions irrigating body areas or organs with the presentcomposition as a means of depleting the local count of CD4+ cells.Exfiltration of CD4+ cells from foreign tissue, such as bone marrow, maybe desirable as a means to providing a pool of CD4+ cells that may betransplanted into a patient to selectively reconstitute CD4+ cells, suchas an individual undergoing chemotherapy and or an HIV-infectedindividual. Depleting CD4+ cells in an organ may also be desirable ifthe organ is to be transplanted, in order to ameliorate complicationssuch as GVHD.

Pharmaceutical Compositions and Routes of Administration

Pharmaceutical compositions comprising Tat and Nef can be formulated andadministered according to well known methods. Tat and Nef, or theirfunctional fragments or derivatives, may be admixed with apharmaceutically acceptable carrier vehicle. Suitable vehicles and theirformulation, inclusive of other human proteins, e.g., human serumalbumin, are described, for example, in Remington's PharmnaceuticalSciences (16th ed., Osol, A., Ed., Mack, Easton Pa. (1980)). In order toform a pharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofTat and Nef proteins, together with a suitable amount of carriervehicle.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts may be formulated foradministration by a variety of routes. The compounds may be delivered byparenteral, inhalation or insufflation (either through the mouth or thenose), topical, oral, or depot administration.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection, repeated injections, or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. Instead of injection, the compounds may be administered asan irrigation fluid used to wash areas or organs of the body.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Formulations and methodology for administering proteins by inhalation ofan individual are known in the art and are described, for example, inU.S. Pat. No. 5,320,094. This patent describes delivery of atherapeutically effective amount of a protein into the lungs of anindividual. The protein is aerosolised and delivered at a regulated flowrate below 30 liters/min. The described methodology is effective todeliver up to 90% of the aerosolised protein into the lungs.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. The compounds may alsobe formulated in rectal compositions such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides.

Topically administered formulations may be in the form of ointments andmay contain pharmaceutically acceptable antibiotics, emollients, andstabilizers. A composition comprising Tat and Nef may be administeredtopically on a surgical apparatus, such as a bandage, gauze, foam, orsurgical instrument.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated or formulated for sustained release by methodswell known in the art. Liquid preparations for oral administration maytake the form of, for example, solutions, syrups or suspensions, or theymaybe presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol or fractionated vegetable oils); and preservatives (e.g., methylor propyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate.

Preferred formulations for oral delivery are described by U.S. Pat. Nos.5,574,018 and 5,428,023. Biologically active conjugates of atherapeutically useful protein are made with vitamin B₁₂(VB₁₂) bycovalently binding the primary (5′) hydroxyl group of the ribose moietyof VB₁₂ to the therapeutic protein. When the resulting conjugate isorally delivered, it binds intrinsic factor (IF) transporter protein inthe gastrointestinal tract and is then taken up through the epitheliuminto the bloodstream, retaining the biological activity of the proteintherapeutic. The conjugates may be orally administered in the presenceof purified IF, resulting in greater absorption.

WO 93/25221 describes compositions formulated for oral delivery,comprising therapeutic proteins contained in microspheres made ofprotein and/or synthetic polymer. The microspheres protect their proteincontents against gastrointestinal proteases and provide controlled andsustained release of their contents. Microspheres can be designed topass through the intestinal epithelium into the blood or lymph, and theymay be targeted to particular cells or organs. Formulations andmethodology useful for targeting orally administered microparticles tovarious organs are described in EP 531,497, for example.

Protein Expression

A particular advantage of the present invention is the provision of Tatand Nef in the form of recombinantly produced proteins, which can beproduced relatively easily and inexpensively. Tat and Nef can beexpressed recombinantly using kiiown methods well known in the art. Tatand Nef can be expressed in a modified form, such as a fusion protein,and can include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, can be added to theN-terminus of a polypeptide to improve stability and persistence in thehost cell, during purification, or during subsequent handling andstorage. Also, peptide moieties can be added to a polypeptide tofacilitate purification. Such regions can be removed prior to finalpreparation of a polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. Such methods are described in many standardlaboratory manuals, such as Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, 2d ed., CSHL Press, NY, Chapters 17.29-17.42 and18.1-18.74.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLE 1 Preparation and Culture of T cells

To obtain a highly enriched culture of CD4+ peripheral blood T cells,250 ml of fresh blood was drawn from a healthy, HIV-negative donor intoheparinized syringes. 31 ml were overlaid on 15 ml of Ficoll-Hypaquecushion solution in each of eight 50 ml conical tubes. The tubes werecentrifuged 40 min at 550×g, and the band of mononuclear cells atop eachcushion was retrieved and pooled into a fresh 50 ml conical tube. Thecells were pelleted at 1,000×g for 10 min and washed 3 times in Earle'sBalanced Salts Solution (EBSS). The fmal pellet was resuspended in 40 mlof RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovineserum (FBS), 2 mM L-glutamine, 1× MEM non-essential amino acid solution,1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycinsulfate. The cell suspension was transferred to a T-75 culture flask andincubated for 2 h at 37° C. in 5% CO₂. Non-adherent cells weretransferred to a 50 ml conical tube, gently pelleted, resuspended inEBSS, and counted. Shutt et al. (1995). 1.25×10⁸ cells were passedthrough a CELLECT column (Biotex Laboratories, Inc., Edmonton, Canada),according to the manufacturer's protocol. CELLECT columns preferentiallyallow flow-through of CD4+ cells by retaining CD8 + and B cells.Alternately, using the same principal of selective adsorption, CD4+cells were purified by magnetic bead separation with kits provided byMiltenyi. The purified CD4+ cells were pelleted, resuspended at a fmalconcentration of 2×10⁶/ml in RPMI 1640 medium supplemented as above, butwith an additional 10% FBS, 10 μg/ml phytohemagglutinin and 10 U/mlIL-2, from which lectin contaminants had been removed. Before use in thechemotaxis experiments, the CD4+ blood cells were grown in this mediumfor a minimum of 72 hrs. Cells of the immortalized SupT1 cell line weremaintained according to methods previously described. Smith et al.(1984); Sylwester et al. (1993).

EXAMPLE 2 Infection with HIV and Obtaining Syncytium-ConditionedSupernatant

HIV-1LAI (previously referred to as HTLV-IIIB) was passed in cellcultures according to methods previously described. Harada et al.(1985). For infection, 10⁷ SupT1 cells were pelleted, resuspended in 1ml of infected culture supernatant containing 500 TCID₅₀ doses ofHIV-1LAI, and incubated for 2 h. Parallel mock infections were performedby resuspending the cells in medium without virus. After incubation,cells were diluted with fresh medium. To obtain syncytium-conditionedsupernatant, 72 to 96 hour post-infected cultures were placed on thestage of an inverted microscope inside a biosafety cabinet, andindividual syncytia were picked with a micropipettor and placed in a 1.5ml screw cap tube with. fresh medium. The syncytia conditioned the mediafor 6 hours, after which time the syncytia were centrifuged and thesupernatant was removed to a fresh tube and stored at −80° C.

EXAMPLE 3 Setting up the Chemotaxis Chamber

Cultures of SupT1 or CD4+ blood cells were grown to a density of 1×10⁶cells/ml, and 200 μl of the culture were seeded onto the centers of22×30 mm THERMANOX® plastic coverslips inside of plastic petri dishes,and were incubated overnight. This step conditioned the surface of thecoverslip, increasing the proportion of motile cells approximatelyfour-fold. Inside the biosafety cabinet, excess medium was removed fromthe coverslip, and the conditioned side of the coverslip was placed ontothe center of the chamber and clamped down. The chamber was theninverted so that cell motility could be measured by their movement overthe conditioned, plastic coverslip. The source and sink wells weresimultaneously filled with the test solutions indicated below. “EBSS” isEarle's Balanced Salt Solution. “Nhs” is normal human serum. “SCM” ismedium conditioned by HIV-induced syncytia. “Cond. Med.” is mediumconditioned by uninfected cells. A glass coverslip was placed over thewells and bridge, first over the sink, then in the direction to thesource, to minimize contamination of the sink solution with the sourcesolution. The chamber was then placed on the stage of the microscope asdescribed below.

EXAMPLE 4 Behavioral Analysis of Cells

The motile behavior of both cells and HIV-induced syncytia were analyzedwith the DIAS software program according to methods previouslydescribed. Soll (1995). The chemotaxis chamber containing the cells andsource and sink solutions was positioned on a Zeiss AXOIVERT 100microscope equipped with long-distance objectives and condenser. Theimage from a COHU videocamera of cells on the bridge of the chamber wasdigitized directly into a computer. Temperature was maintained at 37±1°C. with a thermostatically controlled stage heater. Video images ofsingle cell movements were plotted and digitized by the method of Shuttet al. (1995). The time-dependent location of each cell, or “centroid,”was plotted, as shown in FIG. 2, and instantaneous cellular velocitieswere computed for each centroid by the method of Soll (1995) and Shuttet al. (1995). Other parameters, including directional change, werecomputed according to methods previously described. Soll (1995) andShutt et al. (1995). A cell moving continuously in a straight lineexhibits a directional change value of 0 degrees. Chemotactic index wascomputed as the ‘chemotropism ratio’ of McCutcheon (1944), which is thedistance moved toward the source divided by the total distance moved.The proportion of chemotactically responsive cells (CP+) was computed bydividing the number of cells with a positive chemotactic index by thetotal number of cells and was expressed as a percentage.

EXAMPLE 5 Characterizing the Chemotactic Chamber

Using the described chamber, chemotaxis was analyzed for gradients ofhigh molecular weight molecules. Shutt et al. (1998). The time requiredfor gradient formation is proportional to the molecular weight of thechemoattractant. Tanford (1961). The chamber could then be characterizedby measuring the time required for maximal cellular response to a numberof known chemotactic and chemokinetic agents. Maximum response time wasthen plotted against molecular weight to obtain a plot that could beused to generate a standard curve to estimate the molecular weight of anunknown chemotactic or chemokinetic agent. See FIG. 1A.

EXAMPLE 6 Distinguishing Chemotaxis from Chemokinesis

Since single cell behavior is not assessed in Boyden chambers during theactual process of migration, there has been some concern that someagents interpreted by this method as chemoattractants may in fact beonly chemokinetic agents. Zigmond et al. (1973); Rhodes (1982);Wilkinson (1988). There are computational methods to correct for thisdeficiency, but even when such an analysis is performed, there remainproblems of data interpretation. In addition, because the Boyden chamberprovides only an endpoint of the number of cells on a particular side ofthe chamber, it does not assess the behavior of the test population. Bycontinuously videorecording individual cells at the bridge of thedescribed chemotaxis chamber, the behavior of every cell is continuouslyrecorded and can then be digitized into the computer-assisted DIASmotion analysis system to assess chemokinetic and chemotactic responsesover time. Cells responding chemokinetically but not chemotactically toa stimulatory molecule will exhibit an average instantaneous velocity(I.V.) at optimum concentration significantly higher than that of cellsin buffer, but will never exhibit a chemotatic index (C.I.)significantly different from 0.00, or a percent positive chemotaxisindex (CP+) significantly different from 50%. Cells respondingchemotactically but not chemokinetically to a stimulatory molecule willexhibit an average I.V. that is not significantly different from that inbuffer, but will exhibit an average C.I. and CP+ significantly greaterthan 0.00 and 50%, respectively.

EXAMPLE 7 HIV-induced T cell Syncytia Release Two T CellChemoattractants

Using the single cell chemotaxis chamber, we previously demonstratedthat HIV-induced T cell syncytia release two chemoattractants, oneexerting a maximum effect at approximately 55 minutes and the other atapproximately 115 minutes (Figure B). Shutt et al. (1998). Polyethyleneglycol (PEG)-induced T cell syncytia, which mimic the organization andbehavior of HIV-induced T cell syncytia, do not release eitherattractant, suggesting that both attractants are related to viralinfection. Id. The molecular weights of the two attractants wereestimated to be roughly 30 and 120 kDa (FIG. 1A). The latter attractantwas demonstrated by antibody-blocking experiments to be gp120, andpurified gp120 was demonstrated to be a chemoattractant. Id. Both thehigh and low molecular weight chemoattractants also apparently had achemokinetic effect on peripheral blood T cells. Id.

EXAMPLE 8 Individually, Nef and Tat are Cheynokidetic, but notChemotactic, Stimulators of CD4+ Cells

The absence of chemoattractants in the supernatant of PEG-inducedsyncytia suggested that the chemoattractants released from HIV-inducedsyncytia were encoded by the virus. This proved true for the 120 kDaattractant. Id. We, therefore, tested whether the low molecular weightvirally encoded proteins Rev, p24, Nef and Tat functioned aschemoattractants. Neither Rev, a regulator of viral gene expression, norp24, a capsid protein, stimulated chemoattraction (Table 1) orchemokinesis (Table 2) in twice the time required for these molecules togenerate a gradient in the chamber.

Neither Tat nor Nef functioned as chemoattractants (Table 1). However,both of these latter proteins potently stimulated chemokinesis (Table2). While the proportion of motile cells in buffer was 51% and theaverage instantaneous velocity was 8.3±2.3 μm per mm, the proportion ofmotile cells in a gradient of Tat was 61% and the average instantaneousvelocity was 12.9±3.7 μm per min (Table 2), and the proportion of motilecells in a gradient of Nef was 65% and the average instantaneousvelocity was 11.3±2.8 μm per min. In FIG. 2, centroid tracks arepresented of cells moving in buffer, in a gradient of the low molecularweight chemoattractant of HIV-induced syncytium-conditioned supernatant,in a gradient of Tat and in a gradient of Nef. The centroid tracks ofcells in buffer (FIG. 2A) are short, while those of cells in thesyncytium supernatant gradient (FIG. 2B), the Tat gradient (FIG. 2C) andthe Nef gradient (FIG. 2D) are longer. However, while the majority oftranslocation vectors (75%) point in the direction of increasingsyncytium supernatant chemoattractant (FIG. 2B), the proportion ofvectors pointing in the direction of increasing concentration in the Tatand Nef gradients were 33% and 44%, respectively. Together, theseresults demonstrate that Tat and Nef function as potent chemokineticstimulants of CD4+ cells, but they do not function as chemoattractants.

EXAMPLE 10 A Combination of Tat and Nef Functions as a Chlemoattractantof T Cells

To test whether the chemokinetic stimulation of T cell motility by Tatand Nef was additive or synergistic, cells were analyzed in combinedgradients. The average instantaneous velocity of CD4+ cells in thecombined gradients was similar to that in either the Tat or Nef gradientalone (Table 2). Surprisingly, however, cells now chemotaxed towards thesource of the two gradients. The average chemotactic index in the Tatplus Nef gradients was 0.35±0.43, which was highly similar to thechemotactic index of +0.31±0.49 in a gradient of the lower molecularweight chemoattractant of syncytium supernatant. Both of these C.I.scontrasted markedly with the average C.I.s in buffer (+0.08±0.55), in agradient of Tat alone (+0.06±0.49) or in a gradient of Nef alone(−0.06±0.46) (Table 1). The CP+ in the Tat plus Nef gradients was 80%,which was similar to the CP+ of 78% in a gradient of the lower molecularweight chemoattractant of syncytium supernatant. Both of these CP+scontrasted markedly with the CP+s in buffer (54%), in a gradient of Tatalone (40%) or in a gradient of Nef alone (37%) (Table 1). In FIG. 3, anexample is presented of cell tracks in a combination gradient of Tat andNef. Note that the majority of direction vectors (80%) point in thedirection of increasing concentration and that the tracks are long(compared to tracks in buffer, FIG. 2A), reflecting both chemoattractantand chemokinetic stimulation.

EXAMPLE 11 Nef And Tat Represent the Low Molecular WeightChemoattractant in Medium Conditioned by HIV-Induced T Cell Syncytia

The low molecular weight chemoattractant activity in medium conditionedby HIV-induced T cell syncytia peaked at approximately 55 min, resultingin an estimated molecular weight of approximately 30 kDa. To testwhether Tat and Nef functioned in combination as the low molecularweight chemoattractant, blocking experiments were performed in whichanti-Tat and/or anti-Nef antibody were added to HIV-inducedsyncytium-conditioned medium and the CP+ monitored with time for 55 min.In buffer alone, the CP+ remained close to 50% throughout the 55 minperiod of analysis (FIG. 4A). In a gradient of syncytium supernatant,the CP+ increased to 75% at 55 min (FIG. 4A). In a gradient of Nef orTat, the CP+ remained close to 50% throughout the 55 min period ofanalysis (FIG. 4C), but in a gradient of Nef plus Tat, the CP+ increasedto 80% after 55 min (FIG. 4C). In a gradient of syncytium supernatantcontaining anti-Nef antibody, the CP+ remained close to 50% (FIG. 4 D).An anti-Nef antibody, therefore, blocked the chemotactic activity ofsyncytium supernatant at 55 min. But it did not block chemokineticactivity at 55 mm, which was similar to that of syncytium supernatant inthe absence of antibody, Tat alone, Nef alone or Tat plus Nef (Table 2).Similar results were obtained using an anti-Tat antibody (Table 2).

TABLE 1 Chemotactic response of peripheral blood CD4+ cells 55 min CP+Sink Source N (%) C.I. Cond. Med. Sync. Cond. 27 78% +0.313 ± 0.486Medium (SCM) EBSS + 2% nhs EBSS + 2% nhs 28 54% +0.079 ± 0.554 EBSS + 2%nhs P24 20 60% +0.067 ± 0.434 EBSS + 2% nhs Rev 14 36% +0.060 ± 0.458EBSS + 2% nhs Tat 20 40% +0.060 ± 0.488 EBSS + 2% nhs Nef 19 37% +0.061± 0.462 EBSS + 2% nhs Tat + Nef 40 80% +0.345 ± 0.425 EBSS + 2% nhsSCM + anti-Nef 16 50% +0.016 ± 0.470 Cond. Med. SCM + anti-Tat 20 45%−0.016 ± 0.483 Cond. Med. SCM + anti-Tat + 18 44% −0.007 ± 0.494anti-Nef EBSS + 2% nhs Tat + Nef + anti- 20 40% −0.111 ± 0.495 Tat +anti-Nef

TABLE 2 Chemokinetic response of peripheral blood CD4+ cells N Inst.Vel. Motile Cells Sink Source Analyzed (micron/min) (%) Cond. Med. SCM27 13.3 ± 3.9 87% EBSS + 2% nhs EBSS + 2% 28  8.3 ± 2.3 51% nhs EBSS +2% nhs p24 20  6.5 ± 2.4 52% EBSS + 2% nhs Rev 14  8.5 ± 3.7 55% EBSS +2% nhs Tat 20 12.9 ± 3.7 61% EBSS + 2% nhs Nef 19 11.3 ± 2.8 65% EBSS +2% nhs Tat + Nef 40 12.1 ± 4.1 86% Cond. Med. SCM + anti- 16 12.4 ± 3.967% Nef Cond. Med. SCM + anti- 20 16.9 ± 4.9 84% Tat Cond. Med. SCM +anti- 18 16.2 ± 5.4 79% Tat + anti- Nef EBSS + 2% nhs Tat + Nef + 2012.8 ± 4.1 68% anti-Tat + anti-Nef

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1. A composition comprising Tat and Nef in an effective amount topromote localized accumulation of CD4+ cells, and a compound thatstimulates the differentiation of CD4+ Th0 cells into either Th2 cells.2. The composition of claim 1, wherein said compound stimulatesdifferentiation of CD4+ Th0 cells into Th2 cells, and is selected from acompound consisting of IL-4 or an antibody that is capable of binding toINF-γ.
 3. The composition of claim 1, further comprising an antigen andan adjuvant.